Indicator containing perfluorinated polyurethane membranes for use in optical oxygen sensors

ABSTRACT

A ratiometric method for using optical sensors is provided to measure dissolved oxygen in a fluid sample. The sensor is formulated so as to contain an oxygen permeable membrane of a cured perfluorinated urethane polymer and, incorporated therein, an oxygen-sensitive indicator component and a reference dye component. The emission spectrum from the indicator component is calibrated by comparison with that of the reference dye, which is substantially less sensitive to oxygen-quenching and which has a photodecomposition rate similar to the indicator component. Methods for making and using the membrane and sensor are provided as well.

This application is a division of application Ser. No. 08/120,593 filedon Sep. 13, 1993 now U.S. Pat. No. 5,462,880.

TECHNICAL FIELD

The present invention relates generally to methods of using opticalsensors for measuring dissolved oxygen. More particularly, the inventionrelates to a novel ratiometric method of measuring dissolved oxygenusing an optical sensor system containing an oxygen-permeable membranecomposition of a fluorescent hydrophobic urethane copolymer, an oxygenindicator component and a reference dye component. The inventionadditionally relates to fluorescent polymeric compositions for use in anoptical oxygen sensor, to membranes which may be manufactured therefrom,and to methods of making these sensors and membranes.

BACKGROUND

Chemical sensors are generally known for use in a wide variety of areassuch as medicine, scientific research, industrial applications and thelike. Fiber optic and electrochemical approaches are generally known foruse in situations where it is desired to detect and/or measure theconcentration of a parameter at a remote location without requiringelectrical communication with the remote location. Structures,properties, functions and operational details of fiber optic chemicalsensors can be found in U.S. Pat. No. 4,577,109 to Hirschfeld, U.S. Pat.No. 4,785,814 to Kane, and U.S. Pat. No. 4,842,783 to Blaylock, as wellas Seitz, "Chemical Sensors Based on Fiber Optics," AnalyticalChemistry, Vol. 56, No. 1, January 1984, each of which is incorporatedby reference herein.

Publications such as these generally illustrate that is it known toincorporate a chemical sensor into a fiber optic waveguide, anelectrochemical oxygen sensor or the like, in a manner such that thechemical sensor will interact with the analyte. This interaction resultsin a change in optical properties, which change is probed and detectedthrough the fiber optic waveguide or the like. These optical propertiesof chemical sensor compositions typically involve changes in colors orin color intensities. In these types of systems, it is possible todetect particularly minute changes in the parameter or parameters beingmonitored in order to thereby provide especially sensitive remotemonitoring capabilities.

Chemical sensor compositions that are incorporated at the distal end offiber optic sensors are often configured as membranes that are securedat the distal tip end of the waveguide device or optrode. Sensors ofthis general type are useful in measuring gas concentrations such asoxygen and carbon dioxide, monitoring the pH of a fluid, and the like.Ion concentrations can also be detected, such as potassium, sodium,calcium and metal ions.

A typical fiber optic oxygen sensor positions the sensor material at agenerally distal location with the assistance of various differentsupport means. Support means must be such as to permit interactionbetween the oxygen indicator and the substance being subjected tomonitoring, measurement and/or detection. With certain arrangements, itis desirable to incorporate membrane components into these types ofdevices. Such membrane components must possess certain properties inorder to be particularly advantageous. Many membrane materials have someadvantageous properties but also have shortcomings. Generally speaking,the materials must be biocompatible, hemocompatible for use in thebloodstream, selectively permeable to oxygen molecules, and ofsufficient strength to permit maneuvering of the device without concernabout damage to the oxygen sensor.

It is also desirable to have these membrane materials be photocurable(such that curing is neater, can be done more rapidly, on a smallerscale, and directly on the optical fiber), resistant to shear forces(e.g., as present in a bloodstream), and compatible with differentsubstrates, such that there is a choice of fiber optic materials whichcan be used to fabricate the sensor. It is also preferred, clearly, thata signal of sufficient intensity be produced, such that measurement isas accurate as is reasonably possible. The optical oxygen sensors whichare currently available commercially are frequently inadequate withregard to one or more of the aforementioned criteria.

One principal problem with commonly used chemical indicators is thatthey are photolabile. The radiant energy in light induces photochemicalreactions which hasten the decomposition of the indicators and therebyabbreviate their useful lives. This photodecomposition results in acoordinate signal decay referred to as photodrift.

Various approaches have been used to solve the problem of photodrift.Some environmentally sensitive dyes have a portion of their visiblespectrum which shows either a total environmental insensitivity(isobestic point) or a relative insensitivity. This property can be usedto advantage by ratioing the signal from the environmentally sensitiveportion of a indicator's spectrum to that from the isobestic point. Theratio of the signals should be invariant as the indicator moleculephotodecomposes and the absolute signal value decays. This principle hasbeen employed to ratio the signals obtained from fluorescein whenmeasuring pH.

An alternate method of contending with the problem of photodriftinvolves the use of a separate internal reference dye which isenvironmentally insensitive, but photodecomposes at the same rate as theindicator dye. When an internal reference dye is incorporated into theoptical sensor, the signal from the environmentally sensitive dye may becalibrated by comparison with that from the insensitive dye. Due to thesimilarity of the decay rates of the indicator dye and the referencedye, the ratio of the signals should not vary as the two dyesphotodecompose.

In addition to the problem of photodrift, the photochemical reactionsincident to exposure to light result in the ultimate decomposition ofthe organic dyes used as chemical sensors. The use of a system employinga method of ratioing the signals from indicator and reference dyesextends the intervals between which the sensor needs to be recalibratedto operate with accuracy and precision, i.e., to yield O₂ values whichare within approximately 10% of the true O₂ value.

By irradiating with light of a specific wavelength, more than onespecific wavelength, or a range of wavelengths, which may or may not bethe wavelength of maximum absorption, while measuring the fluorescenceemission at specific wavelengths, which may or may not be the wavelengthof maximum emission intensity, or a range of wavelengths in conjunctionwith specific light filtering devices, so as to discern the fluorescenceemission of the indicator dye from that of the reference dye,calibration of the emission signal of the indicator dye may be effectedby ratioing it to that of the reference dye. This results in a signalratio which is sensitive to the analyte of interest and less sensitiveto the effects of exposure to light (photodecomposition of the signal,photodecomposition of the compound) than a single indicator dye sensorcomposition, and a prolonged useful life of the oxygen sensor.

Organometallic transition complexes which are readily quenchedexperience photodecomposition rates which can be influenced by thesupport means in which they are entrapped for use as a chemical sensor.However, these complexes have no portion of their fluorescence spectrumwhich are environment insensitive. While they are not amenable to use ina single-dye chemical sensor composition ratioing system, they may beemployed in conjunction with a fluorescent organic dye with therequisite decay rate and analyte insensitivity to ratio the emissionsignals therefrom.

The present invention is addressed to a novel ratiometric method ofmeasuring dissolved oxygen in a fluid using optical sensors andfluorescent polymer compositions which have been found to beparticularly suitable for use as membranes and membrane-like componentsin an optical oxygen sensor and which provide for optical sensors whichaddress each of the above-mentioned concerns. That is, optical sensorsas provided herein display excellent adhesion to different types ofsubstrates, eliminating in some cases the need to silanize the substratesurface, provide for superior signal intensity, are quite hemocompatiblerelative to prior art compositions, are rapidly cured with light, areresistant to shear forces such as those present in flowing blood andallow for the ratiometric comparison of signals from environmentallysensitive and insensitive molecules which have the same decay rates.

OVERVIEW OF RELATED ART

The following references relate to one or more aspects of the presentinvention. The first reference relates generally to calibratingtechniques. The subsequent three references relate to optical oxygensensors. The final three references relate to techniques of calibratingfiber optic oxygen sensors.

U.S. Pat. No. 4,792,689 to Peterson describes an improved fiber opticsensor in which a method is provided for correcting for common pathvariation in intensity. The method involves passing two wavelengths oflight through a single sample, one of which results in analyte-sensitivefluorescence emission and the other of which results in analytenonsensitive emission.

U.S. Pat. No. 4,861,727 to Hauenstein et al. describes an oxygen sensorin which oxygen-quenchable luminescent lanthanide complexes are employedas indicators.

U.S. Pat. No. 5,043,286 to Khalil et al. describes a method andapparatus for measuring oxygen concentration in a fluid. The methodinvolves the use of a luminescent, fluorinated platinum or palladiumcomplex as the oxygen indicator.

U.S. Pat. No. 5,057,277 to Mauze et al. describes an organosiliconcomposition for use in chemical sensing. In one embodiment, a siliconematrix having a radiative material such as a ruthenium dye incorporatedtherein is used to determine the concentration of oxygen in an analyte.

U.S. Pat. No. 5,094,959 to Allen et al. describes an oxygen sensor inwhich a single indicator species is used as both the indicator and thereference element.

U.S. Pat. No. 5,094,958 to Klainer and Goswami describes a method for"self-calibrating" an oxygen sensor. In a primary embodiment, the methodinvolves the use of an indicator material which produces two distinctanalyte-specific phosphorescence emissions and two distinctanalyte-nonspecific fluorescence emissions which are ratioed to obtain ameasurement signal that is independent of external factors such asdegradation, leaching, or the like.

U.K. Patent Application No. 2,132,348 to Bacon et al. describes anoxygen sensor in which the gas sensitive indicator component is aluminescent organometallic complex.

Lee et al., Anal. Chem. 59(2):279-283 (1987), discloses optical sensorswhich are internally calibrated by virtue of a single reagent whichgives rise to two luminescence bands, one of which is quenched by oxygenand the other of which is not.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to address theabove-mentioned needs in the art, by providing a method for measuringdissolved oxygen in a fluid using an optical sensor which has improvedphotostability, sensitivity, resolution, solvent resistance, andresistance to shear.

It is another object of the invention to address deficiencies in the artby providing such a method in which the optical sensor is formulatedwith a cured perfluorinated urethane polymer, an oxygen-sensitiveindicator component and a reference dye component, and wherein theoxygen-sensitive indicator is a ruthenium indicator and the referencedye is a perylene derivative.

It is yet a further object of the invention to provide such a methodwherein the apparent quantity of oxygen present in the fluid iscorrected for variation in external factors by determining the ratio ofthe oxygen indicator emission signal to the reference dye emissionsignal.

It is another object of the invention to provide an optical oxygensensor which contains a membrane of a cured perfluorinated urethanepolymer, an oxygen-sensitive indicator component and a reference dyecomponent.

It is still another object of the invention to provide such a sensor inwhich the perfluorinated urethane polymer comprises a perfluorinatedpolyurethane acrylate.

It is a further object of the invention to provide a method of makingsuch an optical oxygen sensor by polymerizing a precursor to aperfluorinated urethane polymer on a fiber optic tip.

It is yet another object of the invention to provide an oxygen-permeablemembrane for use in such a sensor, which comprises a polymeric matrix ofa cured perfluorinated urethane polymer, and, incorporated therein, anoxygen-sensitive indicator component and a reference dye component.

It is a further object of the invention to provide such a membrane inwhich the perfluorinated urethane polymer comprises a perfluorinatedpolyurethane acrylate.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

In one aspect, a method for measuring dissolved oxygen dissolved in afluid is provided, wherein the method comprises:

(a) providing an optical sensor comprising an optical waveguide having adistal end portion for measuring dissolved oxygen in a fluid, e.g., abloodstream or the like, and a proximal end portion for communicationwith means for receiving a signal from the distal end portion, andwherein the distal end portion has an oxygen sensor means comprising across-linked oxygen-permeable membrane of a cured perfluorinatedurethane polymer and, incorporated therein, an indicator composition ofan oxygen indicator and a reference dye, wherein the oxygen indicatorprovides for an analyte-sensitive fluorescence emission signal, andwherein the reference dye provides for an analyte-insensitivefluorescence emission signal;

(b) contacting the fluid sample with the distal end portion of theoptical sensor;

(c) exciting the indicator composition with a radiation of a firstwavelength, to produce an oxygen indicator emission signal at a secondwavelength and a reference dye emission signal at a third wavelength;

(d) calculating the apparent quantity of oxygen present in the fluidsample from the oxygen indicator emission signal; and

(e) correcting the apparent quantity of oxygen present for variationsresulting from external factors, by determining the ratio of the oxygenindicator emission signal to the reference dye emission signal.

In another aspect, an optical sensor is provided for measuring dissolvedoxygen, which comprises an optical waveguide having a distal end portionfor monitoring oxygen within a fluid, e.g., a bloodstream or the like,and a proximal end portion for communication with means for receiving asignal from the distal end portion, and wherein the distal end portionhas an oxygen sensor means comprising a cross-linked oxygen permeablemembrane as summarized above and as will be described in detail below.

In another aspect, a method is provided for making the aforementionedoptical sensor. In a preferred embodiment, the method involvespolymerization of a photocurable polymeric precursor on the fiber optictip, by irradiating the precursor-coated tip through the optical fiber.In another embodiment, polymerization of a perfluorinated urethanepolymer precursor may be effected by contacting the precursor-coated tipwith a cross-linking agent in solution or the like.

In still another aspect, a cross-linked oxygen permeable membrane usefulin optical oxygen sensors is provided, wherein the membrane comprises apolymeric matrix of a cured perfluorinated urethane polymer, and,incorporated therein, an oxygen-sensitive indicator component and areference dye component, as will be described in detail herein. In apreferred embodiment, the perfluorinated urethane polymer is aperfluorinated polyurethane acrylate which comprises a perfluorinatedpolyurethane acrylate precursor cross-linked with a cross-linking agent,the oxygen indicator component is ruthenium indicator and the referencedye component is a perylene derivative.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a generally schematic view of a chemical sensor deviceaccording to the present invention which is incorporated in a fiberoptic oxygen sensor device.

FIG. 2 is an enlarged, detail and generally schematic view of the distalend portion of an oxygen sensor device generally in accordance with FIG.1 and incorporating a monolithic cross-linked fluorocarbon polymeraccording to the present invention.

FIG. 3 is a view similar to FIG. 2 but illustrating a composite membranearrangement.

FIG. 4 shows the excitation and emission spectra of the oxygenindicator, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride(also referred to [Ru(4,7-Ph₂ phen)₃ ]Cl₂ or DPPR) in a perfluorinatedurethane polymer film, as evaluated in Example 3.

FIG. 5 shows the excitation and emission spectra of the reference dye,N,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide, in aperfluorinated urethane polymer film, as evaluated in Example 3.

FIG. 6 shows the composite excitation and emission spectra of the oxygenindicator, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chlorideand the reference dye,N,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide, in asingle perfluorinated urethane polymer film, as evaluated in Example 3.

FIG. 7 show the results of a photo drift study of a typical opticaloxygen sensor, as described in Example 4. Depicted in this Figure is theoxygen signal from tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)chloride, the reference signal fromN,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide, and theratio of the oxygen signal to the reference signal.

FIG. 8 shows the results of a 24-hour in vivo study with uncorrectedoxygen sensor signals as evaluated in Example 5.

FIG. 9 shows the results of a 24-hour in vivo study with oxygen sensorsignals ratioed as evaluated in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions, membranes, sensors and methods ofmanufacture are disclosed and described, it is to be understood thatthis invention is not limited to specific sensor formats, specificmembrane compositions, or particular cross-linking agents or curingprocesses, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms "a," "an" and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "an oxygen indicator" includes mixtures of two or moreoxygen indicators, reference to "a perfluorinated urethane polymer"includes mixtures of such polymers, reference to "a precursor" includesmixtures of two or more precursors, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term "oxygen indicator" is intended to mean an environmentallysensitive, organic and/or organometallic chemical compound which, whenexposed to an appropriate wavelength of light, emits a measurablefluorescence signal which is sensitive to (i.e., quenched by) the oxygento which it is exposed.

The term "reference dye indicator" is used herein to mean an organicand/or organometallic chemical compound which, when exposed to anappropriate wavelength of light, emits a measurable fluorescence signalwhich is substantially insensitive to (i.e., not significantly quenchedby) the analyte of interest, and which displays a photodecompositionrate which is approximately the same as that of a selectedenvironmentally sensitive indicator dye.

The term "polymer" as used herein is intended to include both oligomericand polymeric materials, i.e., compounds which include two or moremonomeric units. Similarly, the term "perfluorinated polyether" linkageis intended to mean a linkage containing at least two perfluorinatedether monomer units, i.e., ether monomer units in which each hydrogenatom normally present has been replaced by a fluorine atom.

The term "urethane" is used herein in its Conventional sense to denoteorganic compounds containing a recurring --O--(CO)--NH-- linkage. Theterm "urethane acrylate polymer" is intended to mean a urethane polymerderived from polymerization of a urethane oligomer having acrylatetermini --O--(CO)--CH═CH₂.

The term "precursor" is used herein to mean a compound which whenpolymerized and/or cross-linked will give rise to a desired polymer. Theterm "photodecomposition" is used herein to refer to the chemicaldecomposition, by photolysis processes, which accompanies theillumination of material. This is distinguishable from "photodecay," thenondestructive process in which a fluorescent molecule in the excitedstate decays to a lower energy state with the concomitant emission oflight.

The "Stern Volmer constant" (K_(sv)) is used herein as it is normallydefined, i.e.,

    Io/I=1+K.sub.sv ([O.sub.2 ])

where "Io" represents the fluorescence at 0% oxygen concentration, "I"represents the measured fluorescence when oxygen is present.

The "drift limit" is used herein as it is normally defined, i.e.,##EQU1## The drift limit thus signifies the fraction of the initialsignal which can be lost through drift and still remain withinapproximately 10% error at 150 mm Hg.

The "drift rate" is used herein as it is normally defined, i.e.,##EQU2## The drift rate is thus the fractional signal a sensor loses perhour during defined burn-in conditions; the units are in % signal lostper hour. "Fractional drift" D_(f) is defined as 1-(DR).

The term "New O₂ /Hour" is the predicted oxygen given by a sensor in 150mm Hg after 1 hour of use, and is calculated as follows: ##EQU3##

In describing chemical compounds herein, the term "lower alkyl" is usedin its conventional sense to mean an alkyl group of 1 to 6 carbon atoms,e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,and the like. "Lower alkylene" refers to a difunctional saturatedbranched or unbranched hydrocarbon chain containing from 1 to 6 carbonatoms, and includes, for example, methylene (--CH₂ --), ethylene (--CH₂CH₂ --), propylene (--CH₂ --CH₂ --CH₂ --), and the like. The term"alkylarylene" refers to a difunctional hydrocarbon moiety containing 1or 2 monocyclic aromatic moieties, either unsubstituted phenyl rings orcontaining one to four substituents such as lower alkyl, halogen, nitro,or the like. "Alkylarylene" linking groups may also contain loweralkylene spacers adjacent the aromatic rings, in which some or all ofthe hydrogen atoms normally present may be replaced with fluorine atoms.

The polymeric compositions which are used to formulate the oxygenpermeable membrane of the invention are cured perfluorinated urethanepolymers; the membrane itself comprises a matrix of such a polymer and,incorporated in the matrix, an oxygen-sensitive indicator component anda reference dye component. The cured perfluorinated urethane polymersare typically perfluorinated urethane polymer precursors cross-linkedwith a cross-linking agent. Generally, such precursors have thestructural formula ##STR1## wherein Ar is a monocyclic aromatic moietyand X is a perfluorinated polyether linkage containing approximately 2to 100, preferably 10 to 50, most preferably 30 to 45, recurringperfluorinated monomer units having the structure (--CF₂ O--), (--CF₂CF₂ O--), or combinations thereof. Hydrophobicity may easily bemodulated by varying the number of perfluorinated ether units containedwithin the moiety X. Preferably, Ar is phenyl, either unsubstituted orsubstituted with one to four substituents which are selected so as notto interfere with polymerization or use of the cured polymer in theoxygen sensor; such substituents include, for example, lower alkyl (C₁-C₆), halogen, nitro, and the like. Hydrophobicity may easily bemodulated by varying the number of perfluorinated ether units containedwithin the moiety X.

The precursor of Formula (I) may be crosslinked using water or anorganic diol HO--R--OH wherein R is a hydrocarbon substituent of about 2to 20 carbon atoms, and in which some or all of the hydrogen atomsnormally present have been replaced with fluorine atoms. Preferably, Ris an alkylene linking group, i.e., an alkylene linking group containingfrom about 1 to 6 carbon atoms, or an alkylarylene linking groupcontaining one or two monocyclic aromatic moieties and, depending on thenumber of aromatic moieties, two or three lower alkylene spacer groups,again, in which some or all of the hydrogen atoms normally present havebeen replaced with fluorine atoms. Exemplary organic diols includebisphenol A and hexafluorobisphenol A.

In a preferred embodiment, the precursor of Formula (I) is converted toa perfluorinated urethane acrylate precursor prior to curing, byreplacing the terminal isocyanate moieties --N═C═O with acrylate termini--NH--COO--(CH₂)_(n) --(CO)--CH═CH₂ where n is typically in the range of1 to about 6. This may be effected by reacting the diisocyanateprecursor (I) with, for example, hydroxymethylmethacrylate (in whichcase n is 1), hydroxyethylmethacrylate (in which case n is 2), or thelike. The perfluorinated urethane acrylate precursor so provided, havingthe structural formula ##STR2## may then be cured in the presence of asuitable photoinitiator or photocatalyst using radiation. In a variationon this embodiment, the diisocyanate-terminated precursor of Formula (I)may be reacted with virtually any compound having a hydroxy terminus anda vinyl terminus, typically containing about 2 to 10 carbon atoms, toprovide a vinyl-terminated precursor and to enable cross-linking.

Suitable photoinitiators for carrying out the cross-linking in theaforementioned case, i.e., to cure the perfluorinated urethane acrylateprecursor of Formula (II), are radical photoinitiators that arewell-known to those skilled in the art. Examples of such photoinitiatorsinclude 2-hydroxy-2,2-dialkyl acetophenones, α-alkoxy deoxybenzoins,α,α-dialkoxy deoxybenzoins, α,α-dialkoxy acetophenones, benzophenones,thioxanthones, benzils, and other compounds identified by H. J. Hagemanet al., "Photoinitiators and Photocatalysts for Various Polymerisationand Crosslinking Processes," in Radiation Curing of Polymers II, ed. D.R. Randell (The Royal Society of Chemistry, 1991), at pp. 46-53, citedsupra. The disclosure of the aforementioned reference is incorporated byreference herein.

In another embodiment, the diisocyanate-terminated precursor of Formula(I) is converted to an epoxy-terminated precursor having the formula##STR3## wherein Ar, X, and n are as defined above. This conversion maybe readily effected by reaction of the precursor of Formula (I) with twoequivalents of a compound having the structural formula ##STR4## (i.e.,glycidol when n is 1). This epoxy-terminated compound may then be curedwith radiation in the presence of a cationic photoinitiator, e.g., asulfonium salt, an organometallic complex such as that manufacturedunder the name Irgacure® by Ciba-Geigy Corporation, or the like.

One of the advantages of fabricating optical oxygen sensors with theaforementioned polymer compositions is that a cross-linking agent isgenerally not required. Conventional systems typically require a veryhigh level of cross-linking agent.

In formulating the oxygen permeable membrane, it is preferred that theabove-described cross-linking reaction occur in the presence of theoxygen-sensitive indicator component and the reference dye componentwhich will then be incorporated into the polymeric matrix which servesas the membrane. The oxygen-sensitive indicator and the reference dyewill generally be physically entrapped within the polymeric matrix, butit may also be covalently bound thereto.

The oxygen-sensitive indicator is typically an inorganic complex whichis a luminescent material quenchable by the oxygen. Examples of suitableoxygen-sensitive indicators useful for oxygen determination may be foundin U.K. Patent No. 2,132,348, cited supra, and include complexes ofruthenium (II), osmium (II), iridium (III), rhodium, rhenium, andchromium (III) with 2,2'-bipyridine, 1,10-phenanthrolene,4,7-diphenyl-1,10-phenanthrolene, 4,7-dimethyl-1,10-phenanthrolene,4,7-disulfonated-diphenyl-1,10-phenanthrolene, 2,2'-bi-2-thiazoline,2,2'-bithiazole, 5-bromo-1,10-phenanthrolene, and5-chloro-1,10-phenanthrolene, and complexes of VO (II), Cu (II),platinum (II), and zinc (II) with porphyrin, etioporphyrin,tetraphenylporphyrin, mesoporphyrin IX dimethylester, protoporphyrin IXdimethylester and octaethylporphyrin. Preferred oxygen-sensitiveindicators for fabricating oxygen optical sensors are rutheniumcomplexes, most preferred istris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride.

The reference dye is typically an organic luminescent material which isrelatively unquenchable by oxygen (i.e., has a Stern Volmer constantK_(sv) which is substantially smaller than that of the oxygen-sensitiveindicator; in general, the K_(sv) of the reference dye should be atleast 0.05 less than that for the oxygen-sensitive indicator), has afluorescence emission spectrum which can be readily discerned from thatof the oxygen indicator dye and has a relatively stable photodrift rate(typically within about 1% when measured at 150 mm Hg) that is closelymatched to that of the oxygen-sensitive indicator. Examples of referencedyes include polynuclear aromatic compounds, such as perylenederivatives, fluorescein and fluorescein derivatives such ascarboxyfluorescein, hydroxypyrene trisulfonic acid, dichlorofluorescein,and the like. Preferred examples of such reference dyes areN,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide andN,N'-bis(2,6-xylidyl)-3,4,9,10-perylenecarboximide (DXP).

The cross-linking reactions which give rise to the oxygen permeablemembrane are preferably carried out on the fiber substrate. In apreferred embodiment, the precursor is photocurable and is cross-linkedon the fiber substrate using radiation transmitted through the fiber.Alternatively, the membrane may be prepared separately and deposited onthe surface of the optical fiber; in such instances, it is typicallynecessary to prime the fiber surface prior to deposition of the sensingmembrane thereonto. An example of a suitable glass primer isγ-methacryloxypropyl trimethoxysilane. Alternatively, the distal tip ofthe fiber may be dipped into a solution of the precursor, theoxygen-sensitive indicator and the reference dye, and taking suitablesteps to cure and cross-link the solution. Once cured, the oxygen sensorthus formed may be cleaned of residual unreacted monomer by soaking inan innocuous solvent such as dimethylsulfoxide or buffer/water. Thepresent invention, however, minimizes the potential for unreactedmonomer and rinsing may be a superfluous step.

The polymer composition--i.e., the cross-linked perfluorinated urethanepolymer--will typically represent on the order of 80 to 99 wt. % of theoxygen permeable membrane, more typically 95 to 99 wt. % of themembrane. Any photoinitiator used will be present at customary levels,typically around 1-2 wt. % of the membrane. The oxygen-sensitiveindicator and reference dye will generally represent on the order of0.03-1.0 wt. % of the membrane.

Perfluorinated urethane polymers and optical sensors formulated withsuch polymers are described in detail in commonly assigned, copendingU.S. patent application Ser. No. 07/911,175, entitled "Cross-Linked GasPermeable Membrane of a Cured Perfluorinated Urethane Polymer, andOptical Gas Sensors Fabricated Therewith," filed 12 Aug. 1992 andincorporated herein by reference. Those skilled in the art who wouldlike further information concerning fiber optic oxygen sensorsformulated with perfluorinated urethane polymers are referred to theaforementioned patent application for additional detail.

FIG. 1 shows a typical fiber optic oxygen sensor arrangement. Theillustrated device 11 includes a light source 12 for directing proberadiation into the device, as well as a light detector 13 for sensingand detecting radiation from the device. Device 11 includes one or moreoptical fibers 14 that are joined to light source 123 and to lightdetector 13 through a suitable junction assembly 15 at a location whichis proximal of the distal end portion 16 of the optical fiber 14. As isgenerally known, each optical fiber 14 includes a core surrounded by acladding or covering.

Distal end portion 16 has a distal tip 17 which is a membrane of across-linked perfluorinated urethane polymer matrix, and, incorporatedtherein, an oxygen-sensitive indicator and a reference dye as describedabove. The oxygen-sensitive indicator enables the matrix to undergo aknown change in color, color intensity or other property, which changeis observed by the light detector 13 in a manner generally known in theart.

With the embodiment illustrated in FIG. 3, a distal end portion 16' hasa distal tip 17'. The tip 17' is a composite membrane suitable formultifunctional monitoring, such as for monitoring pH conditions or thelike and oxygen concentrations. Microparticles 21 of a polymer matrixcomprising a perfluorinated urethane polymer, an oxygen-sensitiveindicator and a reference dye are included within the composite membraneat the distal tip 17'. Also included are other indicator components 22such as fluorescent pH indicators. Both the oxygen sensor microparticles21 and the other indicators 22 are encapsulated within a known type ofoxygen and ion permeable hydrophilic polymer 23 which provide neededsupport for the microparticles therewithin.

Examples of suitable fiber substrate materials include glass, plastic,glass-on-glass and plastic-clad glass fiber waveguides. A criticalcharacteristic of optical fibers is attenuation of the optical signal.Thus, glasses which contain unacceptable levels of transition-metalimpurities when prepared from naturally occurring materials lead to highabsorption losses. High silica fibers of acceptable quality can beprepared from purified starting materials (e.g., silicon tetrachlorideand germanium tetrachloride) using conventional glass-melting techniquesof melting, fining and drawing into fibers. In order to promote adhesionof the membrane to the fiber, the surface of the tip of the fibersubstrate may be silanized, such as with γ-methacryloxypropyltrimethoxysilane as primer, as discussed above.

As noted earlier, the primary utility of the present invention is in thedetection and measurement of dissolved oxygen in the bloodstream.However, the membrane and sensor of the invention may also be used in avariety of other contexts, e.g., for on-line sensing in a flowing fluidstream.

It is to be understood that while the invention has been described inconjunction with preferred specific embodiments thereof, the foregoingdescription, as well as the examples which follow, are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXAMPLE 1

The objective of this example was to prepare a radiation-curablefluoropolyurethane for fabricating an H₂ O and H⁺ impermeable membranewith good elastomeric properties. A difunctional, isocyanate-terminatedfluorinated polyether having an equivalent weight of approximately 1500(Fluorolink™ B, obtained from Ausimont, Morristown, N.J.) was used asthe polymeric precursor. The reactions which were carried out (1)replaced the diisocyanate termini of the precursor with acrylatemoieties, thereby providing a photocurable compound, and (2) cured thislatter acrylate-terminated compound, as follows.

Five g of Fluorolink™ B was weighed out and 0.43 g of dryhydroxyethylmethacrylate (HEMA) (obtained from Aldrich Chemical Company,Inc., Milwaukee, Wis.), which had been stored over 4 Å molecular sieves,was added to the Fluorolink™ B. The reaction was permitted to proceed atroom temperature uncatalyzed. After 1 hour, no apparent exothermoccurred. The reaction was incubated at approximately 20° C. for 18hours. At that time, it was found that the preparation had not cured;accordingly, 5 μl dibutyltin dilaurate (obtained from Air Products andChemicals under the name T-12) catalyst was added, and the preparationbubbled slightly.

The acrylate urethane was found to be soluble in Freon 113trichlorotrifluoroethane; 5 μl of the photoinitiator Irgacure® 500(Ciba-Geigy) was added, and the polymer solution was thus cured under astream of N₂. The structure of the polymer was verified using infraredspectroscopy.

EXAMPLE 2

The objective of this example was to prepare an oxygen sensor bydissolving an oxygen-sensitive indicator and a reference dye in across-linkable, curable polymer matrix that is permeable to oxygen. Asin Example 1, Fluorolink™ B was used as the precursor to the curedperfluorinated urethane acrylate polymer which serves as the primarycomponent of the polymer matrix. In this example, cure was effected withmoisture.

Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride (obtainedfrom Florida International University) was selected as the oxygensensitive indicator andN,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide (DBPI)(Aldrich Chemical Co.) was selected as the reference dye. 1.5 mg each ofDPPR and DBPI was dissolved in 0.2 ml CH₂ Cl₂, and miscibilized in 1.0 gof the perfluorinated prepolymer Fluorolink™ B to which was added 10 μldibutyltin dilaurate as a catalyst. Freon 113 trichlorotrifluoroethane(200 μl) was added and the solution was thoroughly mixed.

The waveguide (Ensign Bickford glass-on-glass, 240 μm, numericalaperture 0.39) was dipped in the polymer/oxygen indicator/reference dyepreparation and allowed to dry at room temperature overnight. The driedsensor was cured at 50° C. in a humidified forced air oven for 18 hours.

EXAMPLE 3

The objective of this experiment was to determine the optimalwavelengths at which to excite an oxygen indicator dye and a referencedye, and at which wavelengths to monitor the fluorescence emissionspectra. Either tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)chloride orN,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide (DBPI)(Aldrich Chemical Co.), or both DPPR and DBPI, was dissolved inmethylene chloride at approximately 0.1 wt. %, miscibilized within theperfluorinated polyurethane prepolymer and cured in the standardfashions, either by moisture curing mechanisms or photocuring theperfluorinated urethane acrylate prepolymer.

Films generated from this polymer-indicator or polymer-reference dyemixture have the following fluorescence spectrum. FIG. 4 shows theexcitation and emission spectra of the oxygen indicator in the urethanefilm. FIG. 5 shows the excitation and emission spectra of the referencedye in the urethane film. FIG. 6 shows the excitation and emissionspectra of the two compounds within the same urethane film. Note thatone can excite both dyes at 485 nm and capture the oxygen emissionsignal at 607 nm and the reference compound signal at 540 nm, all ofwhich are easily deconvolutable using conventional optical filters.Ratioing the signal at 607 nm to that at 540 nm yields the desiredcalibration ratio.

EXAMPLE 4

The purpose of this experiment was to evaluate DBPI as a reference dyefor use with ruthenium oxygen indicator dyes. An optical sensor wasprepared as described in Example 2, incorporating both DPPR and DBPIinto the polymer matrix. The rate of photodrift was measured by excitingboth the indicator and the reference dye at 485 nm, while the oxygenemission signal was captured at 607 nm and the reference compound signalat 540 nm. Ratioing the signal at 607 nm to that at 540 nm yielded thedesired calibration ratio.

The results indicated that the rates of photodecomposition are constantand similar for the indicator dye and the reference dye with exposure tovarious concentrations of dissolved. FIG. 7 shows the results of atypical photo drift study. The unratioed decay rate is substantiallygreater than the ratioed sensor.

The drift rate and useful life of the sensor were compared by monitoringthe signal from the oxygen indicator dye with the ratio of the signalfrom the oxygen indicator dye to the signal from the reference dye. Theresults shown in Table I indicate that the drift rate decreased by morethan half and the useful life is more than doubled for the ratioedsignal relative to the unratioed oxygen signal.

                  TABLE I                                                         ______________________________________                                                       DRIFT    NEW     USEFUL LIFE                                            K.sub.vs *                                                                          RATE     O.sub.2 /HR                                                                           (hours)                                       ______________________________________                                        SIGNAL ONLY                                                                              0.012   1.8%/hr  155   3.33                                        RATIO      0.008   0.8%/hr  152   6.5                                         ______________________________________                                         *Stern Volmer constant, in mm.sup.1                                      

EXAMPLE 5

The object of this experiment was to evaluate the ratiometric method ofthe invention in an in vivo setting. Optical sensors were prepared asdescribed in Example 2. Following standardization against solutionscontaining known amounts of dissolved oxygen, the sensors weresurgically implanted in rabbits. The implanted sensors were continuouslyirradiated at 485 nm (the excitation wavelength) for 24 hours, afterwhich the sensors were removed. The "explant" sensors were againstandardized against solutions of dissolved oxygen as above.

FIGS. 8 and 9 show the results of a 24-hour in vivo study with sensorsunratioed and ratioed, respectively. The drift in the standardizationcurve, and thus the relative instability of the sensor due tophotodecomposition, can be observed in FIG. 8 by comparing thepre-implant signal with the explant signal. By comparison, FIG. 9 showsthat the pre- and post-implant standardization curves are virtuallysuperimposable reflecting the greater stability of the sensor operated.In this particular example, sensor life was extended approximately 110%.

We claim:
 1. A cross-linked oxygen permeable membrane, comprising apolymeric matrix of a cured perfluorinated urethane polymer, and,incorporated therein, an indicator composition of an oxygen indicatorand a reference dye, wherein the oxygen indicator provides for anoxygen-sensitive fluorescence emission signal, and wherein the referencedye provides for an oxygen-insensitive fluorescence emission signal. 2.The membrane of claim 1, wherein the oxygen indicator is selected fromthe group consisting of complexes of ruthenium (II), osmium (II),iridium (III), rhodium, rhenium, and chromium (III) with2,2'-bipyridine, 1,10-phenanthrolene, 4,7-diphenyl-1,10-phenanthrolene,4,7-dimethyl-1,10-phenanthrolene,4,7-disulfonated-diphenyl-1,10-phenanthrolene, 2,2'-bi-2-thiazoline,2,2'-bithiazole, 5-bromo-1,10-phenanthrolene, and5-chloro-1,10-phenanthrolene, and complexes of VO (II), Cu (II),platinum (II), and zinc (II) with porphyrin, etioporphyrin,tetraphenylporphyrin, mesoporphyrin IX dimethylester, protoporphyrin IXdimethylester and octaethylporphyrin.
 3. The membrane of claim 2,wherein the oxygen indicator istris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride and thereference dye is selected from the group consistingofN,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide andN,N'-bis(2,6-xylidyl)-3,4,9,10-perylenecarboximide.
 4. The membrane ofclaim 1, wherein the reference dye is a fluorescent organic compound isselected from the group consisting of fluorescein, carboxyfluorescein,hydroxypyrene trisulfonic acid, dichlorofluorescein,N,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenecarboximide, andN,N'-bis(2,6-xylidyl)-3,4,9,10-perylenecarboximide.
 5. The membrane ofclaim 1, wherein the cured perfluorinated urethane polymer comprises aperfluorinated urethane polymer precursor cross-linked with across-linking agent.
 6. The membrane of claim 5, wherein theperfluorinated urethane polymer precursor has the structural formula##STR5## wherein Ar is a monocyclic aromatic moiety and X is aperfluorinated polyether linkage containing approximately 2 to 100recurring perfluorinated monomer units having the structure (--CF₂ O--),(--CF₂ CF₂ O--), or combinations thereof.
 7. The membrane of claim 6,wherein the cross-linking agent is water.
 8. The membrane of claim 6,wherein the cross-linking agent is a diol.
 9. The membrane of claim 1,wherein the cured perfluorinated urethane polymer comprises aperfluorinated urethane polymer precursor having the structural formula##STR6## wherein Ar is a monocyclic aromatic moiety, X is aperfluorinated polyether linkage containing approximately 2 to 100recurring perfluorinated monomer units having the structure (--CF₂ O--),(--CF₂ CF₂ O--), or combinations thereof, and n is an integer in therange of 0 to 6 inclusive, and wherein the precursor is cured with aphotoinitiator in the presence of radiation.
 10. The membrane of claim1, wherein the cured perfluorinated urethane polymer comprises anepoxy-terminated precursor having the structural formula ##STR7##wherein Ar is a monocyclic aromatic moiety, X is a perfluorinatedpolyether linkage containing approximately 2 to 100 recurringperfluorinated monomer units having the structure (--CF₂ O--), (--CF₂CF₂ O--), or combinations thereof, and n is an integer in the range of 0to 6 inclusive, and wherein the precursor is cured with a photoinitiatorin the presence of radiation.